WO2019239394A1 - Compositions et procédés de détection d'acide nucléique de streptocoque du groupe b - Google Patents

Compositions et procédés de détection d'acide nucléique de streptocoque du groupe b Download PDF

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WO2019239394A1
WO2019239394A1 PCT/IB2019/056814 IB2019056814W WO2019239394A1 WO 2019239394 A1 WO2019239394 A1 WO 2019239394A1 IB 2019056814 W IB2019056814 W IB 2019056814W WO 2019239394 A1 WO2019239394 A1 WO 2019239394A1
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target
seq
rna
dna
hybridizing
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PCT/IB2019/056814
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English (en)
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WO2019239394A8 (fr
Inventor
Barbara L. EATON
Benjamin GROBARCZYK
Yves OZOG
Renaud CLOSE
Laurent FRANZIL
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Gen-Probe Incorporated
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Priority to GB2100233.2A priority Critical patent/GB2590210B/en
Priority to CA3103442A priority patent/CA3103442A1/fr
Priority to AU2019286648A priority patent/AU2019286648B2/en
Priority to CH001571/2020A priority patent/CH716454B1/de
Priority to CN201980051387.4A priority patent/CN112654721A/zh
Publication of WO2019239394A1 publication Critical patent/WO2019239394A1/fr
Publication of WO2019239394A8 publication Critical patent/WO2019239394A8/fr
Priority to AU2023202451A priority patent/AU2023202451B2/en
Priority to AU2023203519A priority patent/AU2023203519B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • GBS Group B Streptococcus
  • Streptococcus agalactiae is a gram-positive bacterium associated with transient colonization of mucosal membranes throughout the body, including the vagina, gastrointestinal tract, and urethra.
  • GBS rarely causes disease in healthy individuals but can cause serious illness in immune compromised patients, elderly individuals, and newborn infants. Of particular concern is neonatal infection caused by vertical transmission during labor and birthing. Transmission from an asymptomatically colonized mother to the neonate can result in early -onset invasive GBS disease, which is the leading cause of sepsis and meningitis in newborns in the United States. Early-onset invasive GBS disease in newborns can result in death or long-term disabilities such as mental retardation and hearing or vision loss. Buchan el al, J. Clin. Microbiol. 53:443-448, 2015.
  • GBS assays having improved sensitivity and/or the potential to protect against isolate variance of a single gene, including, for example, assays that can detect GBS serotypes la, lb, Ic, II, III, IV, V, VI, VII, VIII and IX, including non-hemolytic isolate.
  • the present invention provides a composition for determining the presence or absence of Group B Streptococcus (GBS) in a sample.
  • the composition includes at least one of a first amplification oligomer combination and a second amplification oligomer combination, where
  • the first amplification oligomer combination includes first and second SZP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid, where the first and second .S7/ J -spccific amplification oligomers comprise, respectively, first (A) and second (B) SZP-specific target-hybridizing sequences selected from
  • the second amplification oligomer combination includes first and second CFB- specific amplification oligomers capable of amplifying a target region of a GBS CFB target nucleic acid, where the first and second ⁇ ⁇ Vi-spccific amplification oligomers comprise, respectively, first (A') and second (B') ⁇ ⁇ Vi-spccific target-hybridizing sequences selected from
  • the composition further includes a .S7/ J -spccific detection probe oligomer comprising a SZP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second SZP-specific amplification oligomers.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(a) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(b) and the .S'// J -spccific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the .S'/P-spccific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the .S'/P-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the first CEB-specific target- hybridizing sequence of (II)(a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first CEB-specific target- hybridizing sequence of (II)(a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first CEB-specific target-hybridizing sequence of (II)(a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof. In other embodiments where the CEB-specific target-hybridizing sequences are the target-hybridizing sequences of (II)(a), the first CEB-specific target-hybridizing sequence of (II)(a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof. Particularly suitable first (A') and second (B') CEB-specific target-hybridizing sequences of (II)(a) include
  • the composition further comprises a CEB-specific detection probe oligomer comprising a CEB-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CEB amplicon amplifiable by the first and second CEB-specific amplification oligomers.
  • the first and second CEB-specific target-hybridizing sequences are the target-hybridizing sequences of (II)(b) and the CEB-specific detection probe target- hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CEB-specific target-hybridizing sequences are the target-hybridizing sequences of (II)(d) and the CEB-specific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CEB-specific target-hybridizing sequences are the target-hybridizing sequences of (II)(a) and the f/' Vi-spccific detection probe target- hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof; or the first and second ⁇ ⁇ Vi-spccific target-hybri
  • the CFB- specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the V Y'Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the composition includes both the first and second amplification oligomer combinations.
  • the present invention provides a composition for determining the presence or absence of GBS in a sample, where the composition includes an amplification oligomer combination comprising first and second SZP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid.
  • Particularly suitable first and second SZP-specific amplification oligomers comprise, respectively, first (A) and second (B) SZP-specific target- hybridizing sequences selected from
  • the composition further includes a .S// J -spccific detection probe oligomer comprising a SZP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second SZP-specific amplification oligomers.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (a) and the .SV -spccific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second SIP- specific target-hybridizing sequences are the target-hybridizing sequences of (b) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SZP-specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the SIP- specific detection probe oligomer further includes a non-fluorescent quencher.
  • the composition further includes a second amplification oligomer combination capable of amplifying a target region of a GBS CFB target nucleic acid.
  • the present invention provides a composition for determining the presence or absence of GBS in a sample, where the composition includes an amplification oligomer combination comprising first and second t V'/i-spccific amplification oligomers capable of amplifying a target region of a GBS CFB target nucleic acid.
  • Particularly suitable first and second t V'/i-spccific amplification oligomers comprise, respectively, first (A) and second (B) ( " V'/i-spccific target- hybridizing sequences selected from
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof. In some such embodiments, the first C73 ⁇ 4-specific target-hybridizing sequence of (a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first C73 ⁇ 4-specific target-hybridizing sequence of (a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first ( 7'/i-spccific target- hybridizing sequence of (a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof.
  • Particularly suitable first (A) and second (B) C73 ⁇ 4-specific target-hybridizing sequences of (a) include
  • the composition further includes a / '/'Vi-spccific detection probe oligomer comprising a / '/'Vi-spccific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first and second /’/'Vi-spccific amplification oligomers.
  • the first and second / '/'Vi-spccific target-hybridizing sequences are the target- hybridizing sequences of (b) and the /’/ ' Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB- specific target-hybridizing sequences are the target-hybridizing sequences of (d) and the / '/ ' Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second / '/'Vi-spccific target-hybridizing sequences are the target- hybridizing sequences of (a) and the / '/ ' Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/
  • the / '/ ' Vi-spccific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the / '/ ' Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the composition further includes a second amplification oligomer combination capable of amplifying a target region of a GBS SIP target nucleic acid.
  • the present invention provides an aqueous formulation for the amplification of GBS nucleic acid comprising a composition as above and an organic buffer.
  • the aqueous formulation further includes one or more components selected from a DNA polymerase enzyme, a reverse transcriptase enzyme, a detection probe oligomer, and a bulking agent (e.g. , trehalose, raffinose, or a combination thereof).
  • the aqueous formulation contains inorganic salt at a concentration of 4 mM or less.
  • the present invention provides a reaction mixture for the amplification of GBS nucleic acid comprising an aqueous formulation as above.
  • the present invention provides a dried formulation for the amplification of GBS nucleic acid comprising a composition as above and a bulking agent.
  • the bulking agent is trehalose, raffinose, or a combination thereof.
  • the dried formulation further includes one or more components selected from an inorganic salt, a DNA polymerase enzyme, a reverse transcriptase enzyme, and a detection probe oligomer.
  • the percent mass of the inorganic salt to the mass of the dried formulation is 0.249% or less.
  • the dried formulation is a lyophilized formulation.
  • the present invention provides a reaction mixture for the amplification of GBS nucleic acid, where the reaction mixture is reconstituted with water or an organic buffer from a dried formulation as above.
  • the reaction mixture contains an inorganic salt such as, e.g. , magnesium, potassium, or sodium; in some such variations, the concentration of the inorganic salt is 4 mM or less.
  • the present invention provides a kit for determining the presence or absence of GBS in a sample.
  • the kit includes at least one of a first amplification oligomer combination and a second amplification oligomer combination, where
  • the first amplification oligomer combination includes first and second SZP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid, where the first and second .S7/ J -spccific amplification oligomers comprise, respectively, first (A) and second (B) SZP-specific target-hybridizing sequences selected from
  • the second amplification oligomer combination includes first and second CFB- specific amplification oligomers capable of amplifying a target region of a GBS CFB target nucleic acid, where the first and second f 7 ⁇ //-specific amplification oligomers comprise, respectively, first (A') and second (B') ( " 7 ⁇ //-specific target-hybridizing sequences selected from
  • kits as above where the kit includes the first amplification oligomer combination further includes a S/P-specific detection probe oligomer comprising a SZP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second SZP-specific amplification oligomers.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(a) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(b) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SZP-specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the SIP- specific detection probe oligomer further includes a non-fluorescent quencher.
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (II)(a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (II)(a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first ( 7'Yi-spccific target-hybridizing sequence of (II)(a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (II)(a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof.
  • Particularly suitable first (A') and second (B') C73 ⁇ 4-specific target-hybridizing sequences of (II)(a) include
  • kits as above where the kit includes the first amplification oligomer combination further comprises a f 7 ⁇ Vi-spccific detection probe oligomer comprising a C73 ⁇ 4-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first and second C73 ⁇ 4-specific amplification oligomers.
  • the first and second C73 ⁇ 4-specific target-hybridizing sequences are the target- hybridizing sequences of (II)(b) and the C73 ⁇ 4-specific detection probe target-hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB- specific target-hybridizing sequences are the target-hybridizing sequences of (II)(d) and the CFB- specific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second C73 ⁇ 4-specific target-hybridizing sequences are the target-hybridizing sequences of (II)(a) and the f 7 ⁇ Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof; or the first and second f 7 ⁇ Vi-
  • the detectable label is a fluorescent label and the f 7 ⁇ Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • kits for determining the presence or absence of GBS in a sample as above includes both the first and second amplification oligomer combinations.
  • the present invention provides a kit for determining the presence or absence of GBS in a sample, where the kit includes an amplification oligomer combination comprising first and second SZP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid.
  • amplification oligomer combination comprising first and second SZP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid.
  • Particularly suitable first and second .SV -spccific amplification oligomers comprise, respectively, first (A) and second (B) SVP-specific target- hybridizing sequences selected from
  • the kit further includes a SZP-specific detection probe oligomer comprising a SIP- specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second SZP-specific amplification oligomers.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (a) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second SZP-specific target- hybridizing sequences are the target-hybridizing sequences of (b) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SZP-specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the S/P-specific detection probe oligomer further includes a non-fluorescent quencher.
  • the kit further includes a second amplification oligomer combination capable of amplifying a target region of a GBS CFB target nucleic acid.
  • the present invention provides a kit for determining the presence or absence of GBS in a sample, where the kit includes an amplification oligomer combination comprising first and second t V'/i-spccific amplification oligomers capable of amplifying a target region of a GBS CFB target nucleic acid.
  • Particularly suitable first and second C73 ⁇ 4-specific amplification oligomers comprise, respectively, first (A) and second (B) ⁇ ⁇ Vi-spccific target- hybridizing sequences selected from
  • the first f '/'Vi-spccific target-hybridizing sequence of (a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first f 7' Vi-spccific target-hybridizing sequence of (a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof. In other embodiments, the first ( 7'Vi-spccific target- hybridizing sequence of (a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof.
  • Particularly suitable first (A) and second (B) C73 ⁇ 4-specific target-hybridizing sequences of (a) include
  • the kit further includes a ⁇ ⁇ Vi-spccific detection probe oligomer comprising a ("/'Vi-spccific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first and second C73 ⁇ 4-specific amplification oligomers.
  • the first and second C73 ⁇ 4-specific target-hybridizing sequences are the target- hybridizing sequences of (b) and the C73 ⁇ 4-specific detection probe target-hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB- specific target-hybridizing sequences are the target-hybridizing sequences of (d) and the ("/'Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB-specific target-hybridizing sequences are the target- hybridizing sequences of (a) and the ("/'Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof; or the first and second C73 ⁇ 4-specific target-hybridizing sequences are the
  • the (’/'Vi-spccific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the ("/'Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the kit further includes a second amplification oligomer combination capable of amplifying a target region of a GBS SIP target nucleic acid.
  • the present invention provides a method for determining the presence or absence of GBS in a sample.
  • the method includes
  • the first amplification oligomer combination includes first and second SZP-specific amplification oligomers for amplifying a target region of a GBS SIP target nucleic acid, where the first and second SZP-specific amplification oligomers comprise, respectively, first (A) and second (B) SZP-specific target-hybridizing sequences selected from
  • the second amplification oligomer combination includes first and second CFB- specific amplification oligomers for amplifying a target region of a GBS CFB target nucleic acid, where the first and second ⁇ ⁇ Vi-spccific amplification oligomers comprise, respectively, first (A') and second (B') ⁇ ⁇ Vi-spccific target-hybridizing sequences selected from
  • the method includes contacting the sample with both the first and second amplification oligomer combinations.
  • the method is a multiplex method comprising contacting the sample with both the first and second amplification oligomer combinations within the same reaction mixture.
  • the detecting step includes contacting the in vitro nucleic acid amplification reaction with a .S7/ J -spccific detection probe oligomer comprising a SZP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second SZP-specific amplification oligomers.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(a) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second SZP-specific target-hybridizing sequences are the target-hybridizing sequences of (I)(b) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SZP-specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the SIP- specific detection probe oligomer further includes a non-fluorescent quencher.
  • the first CGB-specific target- hybridizing sequence of (II)(a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first ( V'/i-spccific target- hybridizing sequence of (II)(a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first ( 7'/i-spccific target-hybridizing sequence of (II)(a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first OMi-spccific target-hybridizing sequence of (II)(a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof.
  • Particularly suitable first (A 1 ) and second (B') G/'Vi-spccific target-hybridizing sequences of (II)(a) include (i) (A') SEQ ID NO: 12, or an RNA equivalent or DNA/RNA chimeric thereof, and (B') SEQ ID NO: 13, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the detecting step includes contacting the in vitro nucleic acid amplification reaction with a ⁇ ⁇ Vi-spccific detection probe oligomer comprising a C73 ⁇ 4-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first and second C73 ⁇ 4-specific amplification oligomers.
  • the first and second C73 ⁇ 4-specific target-hybridizing sequences are the target- hybridizing sequences of (II)(b) and the C73 ⁇ 4-specific detection probe target-hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB- specific target-hybridizing sequences are the target-hybridizing sequences of (II)(d) and the CFB- specific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second ⁇ ⁇ Vi-spccific target-hybridizing sequences are the target-hybridizing sequences of (II)(a) and the V Y'Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof; or the first and second V
  • the detectable label is a fluorescent label and the V Y'Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the detecting step is performed in real time.
  • the in vitro nucleic acid amplification reaction is a PCR amplification reaction (e.g. , a real-time PCR amplification reaction).
  • the detecting step includes contacting the in vitro nucleic acid amplification reaction with (i) a SIP- specific detection probe oligomer comprising a .S7/ J -spccific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second .S7/ J -spccific amplification oligomers, and (ii) a C73 ⁇ 4-specific detection probe oligomer comprising a C73 ⁇ 4-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first
  • the present invention provides a method for determining the presence or absence of GBS in a sample, where the method includes
  • first and second STP-specific amplification oligomers for amplifying a target region of a GBS SIP target nucleic acid, where the first and second STP-specific amplification oligomers comprise, respectively, first (A) and second (B) SIP- specific target-hybridizing sequences selected from
  • the detecting step comprises contacting the in vitro nucleic acid amplification reaction with a STP-specific detection probe oligomer comprising a STP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by the first and second STP-specific amplification oligomers.
  • the first and second STP-specific target-hybridizing sequences are the target-hybridizing sequences of (a) and the STP-specific detection probe target-hybridizing sequence is SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first and second .S7/ J -spccific target-hybridizing sequences are the target-hybridizing sequences of (b) and the SZP-specific detection probe target-hybridizing sequence is SEQ ID NO: 11, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SIP- specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the S/P-specific detection probe oligomer further includes a non-fluorescent quencher.
  • the detecting step is performed in real time.
  • the in vitro nucleic acid amplification reaction is a PCR amplification reaction (e.g., a real-time PCR amplification reaction).
  • the method further includes contacting the sample with a second amplification oligomer combination comprising first and second C/ ⁇ Vi-spccific amplification oligomers for amplifying a target region of a GBS CFB target nucleic acid, where, at the amplification step, any GBS CFB target nucleic acid, if present in the sample, is used as a template for generating an amplicon corresponding to the CFB target region, and where the detecting step includes detecting the presence or absence of the amplicon corresponding to the CFB target region.
  • the present invention provides a method for determining the presence or absence of GBS in a sample, where the method includes
  • first and second C73 ⁇ 4-specific amplification oligomers for amplifying a target region of a GBS CFB target nucleic acid, where the first and second G/' Vi-spccific amplification oligomers comprise, respectively, first (A) and second (B)
  • the first V Y'Yi-spccific target-hybridizing sequence of (a) includes at least the sequence of SEQ ID NO:28, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first C73 ⁇ 4-specific target-hybridizing sequence of (a) is contained in the sequence of SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof; in some such variations, the first C73 ⁇ 4-specific target-hybridizing sequence of (a) is SEQ ID NO: 12 or SEQ ID NO: 14, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the first ( Y'/i-spccific target- hybridizing sequence of (a) is SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof.
  • Particularly suitable first (A) and second (B) C73 ⁇ 4-specific target-hybridizing sequences of (a) include
  • the detecting step includes contacting the in vitro nucleic acid amplification reaction with a ⁇ ⁇ Vi-spccific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by the first and second ⁇ ⁇ Vi-spccific amplification oligomers.
  • the first and second V Y'Vi-spccific target-hybridizing sequences are the target- hybridizing sequences of (b) and the V V'Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second CFB- specific target-hybridizing sequences are the target-hybridizing sequences of (d) and the V Y'Vi-spccific detection probe target-hybridizing sequence is SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof;
  • the first and second V Y'/i-spccific target-hybridizing sequences are the target- hybridizing sequences of (a) and the V Y'/i-spccific detection probe target-hybridizing sequence is SEQ ID NO:22 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof; or the first and
  • the ( 7'/i-spccific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the ⁇ ⁇ Vi-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the detecting step is performed in real time.
  • the in vitro nucleic acid amplification reaction is a PCR amplification reaction (e.g., a real-time PCR
  • the method further includes contacting the sample with a second amplification oligomer combination comprising first and second .SV -spccific amplification oligomers for amplifying a target region of a GBS SIP target nucleic acid, where, at the amplification step, any GBS SIP target nucleic acid, if present in the sample, is used as a template for generating an amplicon corresponding to the SIP target region, and where the detecting step comprises detecting the presence or absence of the amplicon corresponding to the SIP target region.
  • the method determines the presence or absence of any of GBS serotypes la, lb, Ic, II, III, IV, V, VI, VII, VIII, and IX. In some such embodiments, the method further determines the presence or absence of a non-hemolytic strain of GBS.
  • the present invention provides a detection probe oligomer.
  • the detection probe oligomer is a SVP-specific detection probe oligomer comprising a SVP-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a SIP amplicon amplifiable by a first amplification oligomer combination comprises first and second SVP-specific amplification oligomers capable of amplifying a target region of a GBS SIP target nucleic acid, wherein the first and second SVP-specific amplification oligomers comprise, respectively, first (A) and second (B) SVP-specific target-hybridizing sequences selected from the group consisting of
  • the SVP-specific detection probe target-hybridizing sequence is selected from (a) SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof, and (b) SEQ ID NO: 11 , or an RNA equivalent or DNA/RNA chimeric thereof.
  • the SVP-specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the SIP- specific detection probe oligomer further includes a non-fluorescent quencher.
  • the detection probe oligomer is a V Y ' Yi-spccific detection probe oligomer comprising a V Y ' Yi-spccific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a CFB amplicon amplifiable by a second amplification oligomer combination comprises first and second ⁇ ⁇ Vi-spccific amplification oligomers capable of amplifying a target region of a GBS CFB target nucleic acid, wherein the first and second C73 ⁇ 4-specific amplification oligomers comprise, respectively, first (A 1 ) and second (B') ⁇ ⁇ Vi-spccific target-hybridizing sequences selected from
  • the V Y ' Yi-spccific detection probe target-hybridizing sequence is selected from (a) SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof, (b) SEQ ID NO:25, or an RNA equivalent or DNA/RNA chimeric thereof, (c) SEQ ID NO:22, or an RNA equivalent or DNA/RNA chimeric thereof, and (d) SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the CFB- specific detection probe oligomer further includes a detectable label such as, for example, a fluorescent or chemiluminescent label.
  • the detectable label is a fluorescent label and the V Y'/i-spccific detection probe oligomer further includes a non-fluorescent quencher.
  • the present invention provides a composition comprising a SIP- specific detection probe oligomer and a V Y ' Vi-spccific detection probe oligomer as above.
  • the present invention provides an aqueous formulation for the detection of GBS nucleic acid comprising (1) a SYP-specific detection probe oligomer and/or a CFB- specific detection probe oligomer as above and (2) an organic buffer.
  • the aqueous formulation further includes one or more components selected from a surfactant (e.g., polyethylene glycol mono [4-(l,l,3,3-tetramethylbutyl) phenyl] ether, polysorbate 20, or a combination thereof), a DNA polymerase enzyme, a reverse transcriptase enzyme, at least one amplification oligomer, and a bulking agent (e.g., trehalose, raffinose, or a combination thereof).
  • the surfactant is a non-linear surfactant such as, for example, polysorbate 20.
  • the aqueous formulation contains inorganic salt at a concentration of 4 mM or less.
  • the present invention provides a reaction mixture for the detection of GBS comprising an aqueous formulation as above.
  • the present invention provides a dried formulation for the detection of GBS nucleic acid comprising (1) a .S'/ -spccific detection probe oligomer and/or a C73 ⁇ 4-specific detection probe oligomer as above and (2) a bulking agent.
  • the bulking agent is trehalose, raffinose, or a combination thereof.
  • the dried formulation further includes one or more components selected from an inorganic salt, a DNA polymerase enzyme, a reverse transcriptase enzyme, at least one amplification oligomer, and a surfactant (e.g., polyethylene glycol mono [4-(l,l,3,3-tetramethylbutyl) phenyl] ether, polysorbate 20, or a combination thereof).
  • a surfactant e.g., polyethylene glycol mono [4-(l,l,3,3-tetramethylbutyl) phenyl] ether, polysorbate 20, or a combination thereof.
  • the percent mass of the inorganic salt to the mass of the dried formulation is 0.249% or less.
  • the surfactant is a non-linear surfactant such as, for example, polysorbate 20.
  • the dried formulation is a lyophilized formulation.
  • the present invention provides a reaction mixture for the detection of GBS, where the reaction mixture is reconstituted with water and an organic buffer from a dried formulation as above.
  • the reaction mixture contains an inorganic salt such as, e.g. , magnesium, potassium, or sodium; in some such variations, the concentration of the inorganic salt is 4 mM or less.
  • nucleic acid as used herein is understood to represent one or more nucleic acids.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • Consisting essentially of means that additional component(s), composition(s) or method step(s) that do not materially change the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include the ability to detect a Group B Streptococcus (GBS) nucleic acid sequence present in a sample with specificity that distinguishes the GBS nucleic acid from other known pathogens, optionally at a sensitivity that can detect the bacterium present in a sample at a concentration of about 100 CFU/ml, and, optionally within about 60 minutes and/or within about 40 cycles from the beginning of an amplification reaction when a cycled amplification reaction is used.
  • GBS Group B Streptococcus
  • Sample includes any specimen that may contain GBS or components thereof, such as nucleic acids or fragments of nucleic acids.
  • Samples include "biological samples” which include any tissue or material derived from a living or dead human that may contain GBS or target nucleic acid derived therefrom, including, e.g., vaginal swab samples, cervical brush samples, respiratory tissue or exudates such as bronchoscopy, bronchoalveolar lavage (B AL) or lung biopsy, sputum, saliva, peripheral blood, plasma, serum, lymph node, gastrointestinal tissue, feces, urine, semen or other body fluids or materials.
  • biological samples include any tissue or material derived from a living or dead human that may contain GBS or target nucleic acid derived therefrom, including, e.g., vaginal swab samples, cervical brush samples, respiratory tissue or exudates such as bronchoscopy, bronchoalveolar lavage (B AL) or lung biopsy, sputum, saliva
  • the biological sample may be treated to physically or mechanically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents and the like, which are used to prepare, using standard methods, a biological sample for analysis.
  • samples may include processed samples, such as those obtained from passing samples over or through a filtering device, or following centrifugation, or by adherence to a medium, matrix, or support.
  • Nucleic acid and “polynucleotide” refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together to form a polynucleotide, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof.
  • a nucleic acid "backbone” may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds ("peptide nucleic acids” or PNA; PCT Publication No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
  • Sugar moieties of a nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions, e.g., T methoxy or 2' halide substitutions.
  • Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al, ed., 11 th ed., 1992), derivatives of purines or pyrimidines (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position, purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, 0 6 -methylguanine, 4-thio- pyrimidines, 4-amino-pyrimidines, 4-dimethyl
  • Nucleic acids may include one or more "abasic" residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Patent No. 5,585,481).
  • a nucleic acid may comprise only conventional RNA or DNA sugars, bases and linkages, or may include both conventional components and substitutions (e.g. , conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs).
  • Nucleic acid includes "locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41).
  • LNA locked nucleic acid
  • Embodiments of oligomers that may affect stability of a hybridization complex include PNA oligomers, oligomers that include 2'- methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric associations of a hybridization complex, including oligomers that contain charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates).
  • 5-methylcytosines may be used in conjunction with any of the foregoing backbones/sugars/linkages including RNA or DNA backbones (or mixtures thereof) unless otherwise indicated.
  • a "nucleotide” as used herein is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (also referred to herein as “nucleobase”).
  • the 5- carbon sugar found in RNA is ribose.
  • the 5-carbon sugar is 2'-deoxyribose.
  • the term also includes analogs of such subunits, such as a methoxy group at the 2' position of the ribose (also referred to herein as "2'-0-Me" or "2'-methoxy”).
  • RNA and DNA equivalents are meant RNA and DNA molecules having essentially the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. The differences between RNA and DNA equivalents do not contribute to differences in homology because the equivalents have the same degree of complementarity to a particular sequence.
  • DNA/RNA chimeric is meant a nucleic acid comprising both DNA and RNA nucleotides. Unless the context clearly dictates otherwise, reference to a GBS nucleic acid includes GBS RNA and DNA equivalents and DNA/RNA chimerics thereof.
  • a "target nucleic acid” as used herein is a nucleic acid comprising a target sequence to be amplified.
  • Target nucleic acids may be DNA or RNA, and may be either single -stranded or double-stranded.
  • the target nucleic acid may include other sequences besides the target sequence, which may not be amplified.
  • target sequence refers to the particular nucleotide sequence of the target nucleic acid that is to be amplified and/or detected.
  • the "target sequence” includes the complexing sequences to which oligonucleotides (e.g., priming oligonucleotides and/or promoter oligonucleotides) complex during an amplification processes (e.g., PCR, TMA).
  • oligonucleotides e.g., priming oligonucleotides and/or promoter oligonucleotides
  • target sequence will also refer to the sequence complementary to the "target sequence” as present in the target nucleic acid.
  • target nucleic acid is originally double-stranded, the term “target sequence” refers to both the sense (+) and antisense (-) strands.
  • Target-hybridizing sequence or “target-specific sequence” is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence.
  • the target-hybridizing sequences are configured to specifically hybridize with a target nucleic acid sequence.
  • Target-hybridizing sequences may be 100% complementary to the portion of the target sequence to which they are configured to hybridize, but not necessarily.
  • Target-hybridizing sequences may also include inserted, deleted and/or substituted nucleotide residues relative to a target sequence. Less than 100% complementarity of a target-hybridizing sequence to a target sequence may arise, for example, when the target nucleic acid is a plurality strains within a species, such as would be the case for an oligomer configured to hybridize to various serotypes of GBS. It is understood that other reasons exist for configuring a target-hybridizing sequence to have less than 100% complementarity to a target nucleic acid.
  • target a sequence refers to a process whereby an oligonucleotide hybridizes to a target sequence in a manner that allows for amplification and detection as described herein.
  • the oligonucleotide is complementary with the targeted GBS nucleic acid sequence and contains no mismatches.
  • the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted GBS nucleic acid sequence.
  • the oligomer specifically hybridizes to the target sequence.
  • amplification oligomers that are configured to generate a specified amplicon from a target sequence have polynucleotide sequences that hybridize to the target sequence and can be used in an amplification reaction to generate the amplicon.
  • oligonucleotides that are configured to specifically hybridize to a target sequence have a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.
  • oligonucleotide is designed to have a polynucleotide sequence that could target a sequence of the referenced GBS target region. Such an oligonucleotide is not limited to targeting that sequence only, but is rather useful as a composition, in a kit, or in a method for targeting a GBS target nucleic acid.
  • the oligonucleotide is designed to function as a component of an assay for amplification and detection of GBS from a sample, and therefore is designed to target GBS in the presence of other nucleic acids commonly found in testing samples.
  • “Specifically hybridize to” does not mean exclusively hybridize to, as some small level of hybridization to non-taiget nucleic acids may occur, as is understood in the art. Rather, “specifically hybridize to” means that the oligonucleotide is configured to function in an assay to primarily hybridize the target so that an accurate detection of target nucleic acid in a sample can be determined.
  • region refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid.
  • the term “region” may be used refer to the smaller promoter portion.
  • the term “region” may be used to refer to a smaller area of the nucleic acid, wherein the smaller area is targeted by one or more oligonucleotides of the present disclosure.
  • the term region may be used to refer to the smaller nucleotide sequence identified for hybridization by the target-hybridizing sequence of a probe.
  • Oligomer refers to a nucleic acid of generally less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 2 to 5 nt and an upper limit of about 500 to 900 nt. Some particular embodiments are oligomers in a size range with a lower limit of about 5 to 15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50 to 600 nt, and other particular embodiments are in a size range with a lower limit of about 10 to 20 nt and an upper limit of about 22 to 100 nt. Oligomers may be purified from naturally occurring sources, but may be synthesized by using any well-known enzymatic or chemical method. The term
  • oligonucleotide does not denote any particular function to the reagent; rather, it is used generically to cover all such reagents described herein.
  • An oligonucleotide may serve various different functions. For example, it may function as a primer if it is specific for and capable of hybridizing to a complementary strand and can further be extended in the presence of a nucleic acid polymerase; it may function as a primer and provide a promoter if it contains a sequence recognized by an RNA polymerase and allows for transcription (e.g.
  • a T7 Primer may function to detect a target nucleic acid if it is capable of hybridizing to the target nucleic acid, or an amplicon thereof, and further provides a detectible moiety (e.g., an acridinium-ester compound).
  • Oligomers may be referred to by a functional name (e.g. , capture probe, primer or promoter primer) but those skilled in the art will understand that such terms refer to oligomers.
  • an oligonucleotide "substantially corresponding to" a specified reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence in that it would hybridize with the same target nucleic acid sequence under stringent hybridization conditions.
  • substantially corresponding oligonucleotides can vary from a reference sequence and still hybridize to the same target nucleic acid sequence.
  • a first nucleic acid corresponding to a second nucleic acid includes the RNA or DNA equivalent thereof as well as DNA/RNA chimerics thereof, and includes the complements thereof, unless the context clearly dictates otherwise.
  • This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe or primer and its target sequence; thus, in certain embodiments, an oligonucleotide "substantially corresponds" to a reference nucleic acid sequence if these percentages of base identity or complementarity are from 100% to about 80%, preferably from 100% to about 85%, or more preferably from 100% to about 90% or from 100% to about 95%.
  • an oligonucleotide substantially corresponds to a reference nucleic acid sequence if this number of nucleobase substitutions or mismatches is up to four, preferable up to three, or more preferably up to two or up to one substitution(s) or mismatch(es) ( i.e . , from zero to four, preferably from zero to three, or more preferably from zero to two or from zero to one, inclusive).
  • a region of a nucleic acid or amplified nucleic acid can be referred to herein as corresponding to a reference nucleic acid sequence.
  • One skilled in the art will understand the various modifications to the hybridization conditions that might be required at various percentages of complementarity to allow hybridization to a specific target sequence without causing an unacceptable level of non-specific hybridization.
  • the phrase "or its complement, or an RNA equivalent or DNA/RNA chimeric thereof," with reference to a DNA sequence includes (in addition to the referenced DNA sequence) the complement of the DNA sequence, an RNA equivalent of the referenced DNA sequence, an RNA equivalent of the complement of the referenced DNA sequence, a DNA/RNA chimeric of the referenced DNA sequence, and a DNA/RNA chimeric of the complement of the referenced DNA sequence.
  • the phrase "or its complement, or a DNA equivalent or DNA/RNA chimeric thereof," with reference to an RNA sequence includes (in addition to the referenced RNA sequence) the complement of the RNA sequence, a DNA equivalent of the referenced RNA sequence, a DNA equivalent of the complement of the referenced RNA sequence, a DNA/RNA chimeric of the referenced RNA sequence, and a DNA/RNA chimeric of the complement of the referenced RNA sequence.
  • An "amplification oligonucleotide” or “amplification oligomer” is an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction, e.g., serving as a primer or and promoter-primer.
  • Particular amplification oligomers contain at least about 10 contiguous bases, and optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases, that are complementary to a region of the target nucleic acid sequence or its complementary strand.
  • the contiguous bases may be at least about 80%, at least about 90%, or completely complementary to the target sequence to which the amplification oligomer binds.
  • amplification oligomers include all whole and rational numbers within the range (e.g., 92% or 98.377%).
  • Particular amplification oligomers are about 10 to about 60 bases long and optionally may include modified nucleotides.
  • a "primer” is an oligomer that hybridizes to a template nucleic acid and has a 3' end that is extended by polymerization.
  • a primer may be optionally modified, e.g., by including a 5' region that is non-complementary to the target sequence. Such modification can include functional additions, such as tags, promoters, or other non-target-specific sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.
  • a primer modified with a 5' promoter sequence is referred to herein as a "promoter-primer.”
  • promoter-primer A person of ordinary skill in the art of molecular biology or biochemistry will understand that an oligomer that can function as a primer can be modified to include a 5' promoter sequence and then function as a promoter-primer, and, similarly, any promoter-primer can serve as a primer with or without its 5' promoter sequence.
  • a promoter-primer modified to incorporate a 3' blocked end is referred to herein as a "promoter provider,” which is capable of hybridizing to a target nucleic acid and providing an upstream promoter sequence that serves to initiate transcription, but does not provide a primer for oligo extension.
  • Non-target-specific sequence or “non-target-hybridizing sequence” as used herein refers to a region of an oligomer sequence, wherein said region does not stably hybridize with a target sequence under standard hybridization conditions. Oligomers with non-target-specific sequences include, but are not limited to, promoter primers and molecular beacons.
  • Nucleic acid amplification refers to any in vitro procedure that produces multiple copies of a target nucleic acid sequence, or its complementary sequence, or fragments thereof (i.e., an amplified sequence containing less than the complete target nucleic acid).
  • Examples of nucleic acid amplification procedures include transcription associated methods, such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) and others (e.g., U.S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase- mediated amplification (e.g., U.S. Patent No.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand- displacement amplification
  • Amplification may be linear or exponential.
  • Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as QB-replicase.
  • PCR amplification uses DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of the two complementary strands of DNA or cDNA.
  • amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation.
  • Helicase-dependent amplification uses a helicase to separate the two strands of a DNA duplex generating single-stranded templates, followed by hybridization of sequence-specific primers hybridize to the templates and extension by DNA polymerase to amplify the target sequence.
  • SDA uses a primer that contains a recognition site for a restriction endonuclease that will nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps.
  • Transcription associated amplification uses a DNA polymerase, an RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, a promoter-containing
  • oligonucleotide and optionally may include other oligonucleotides, to ultimately produce multiple RNA transcripts from a nucleic acid template (described in detail in, e.g., U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian el al. ; U.S. Patent No. 5,437,990 to Burg el al. ; PCT Publication Nos. WO 88/01302 and WO 88/10315 (Gingeras et al.); U.S. Patent No. 5,130,238 to Malek el al.; U.S. Patent Nos. 4,868,105 and 5,124,246 to Urdea et al.
  • a nucleic acid template described in detail in, e.g., U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian el al. ; U.S. Patent No. 5,437,990 to Burg el al.
  • Threehold cycle is a measure of the emergence time of a signal associated with amplification of target, and is generally lOx standard deviation of the normalized reporter signal.
  • amplification reaches the "threshold cycle,” generally there is considered to be a positive amplification product of a sequence to which the probe binds.
  • the identity of the amplification product can then be determined through methods known to one of skill in the art, such as gel electrophoresis, nucleic acid sequencing, and other such analytical procedures.
  • amplicon or “amplification product” is meant a nucleic acid molecule generated in a nucleic acid amplification reaction and which is derived from a target nucleic acid.
  • An amplicon or amplification product contains a target nucleic acid sequence that may be of the same or opposite sense as the target nucleic acid.
  • RFU relative fluorescence unit
  • Detection probe oligomer refers to an oligomer that hybridizes specifically to a target sequence, including an amplified sequence, under conditions that promote nucleic acid hybridization, for detection of the target nucleic acid. Detection may either be direct (/. e. , probe hybridized directly to the target) or indirect (/. e. , a probe hybridized to an intermediate structure that links the probe to the target). Detection probes may be DNA, RNA, analogs thereof or combinations thereof (e.g. , DNA/RNA chimerics), and they may be labeled or unlabeled. Detection probes may further include alternative backbone linkages such as, e.g.
  • a probe's target sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically.
  • a detection probe may include target-specific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences which will confer a desired secondary or tertiary structure, such as a hairpin structure, which can be used to facilitate detection and/or amplification (see, e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412).
  • Probes of a defined sequence may be produced by techniques known to those of ordinary skill in the art, such as by chemical synthesis, and by in vitro or in vivo expression from recombinant nucleic acid molecules.
  • hybridization or “hybridize” is meant the ability of two completely or partially complementary nucleic acid strands to come together under specified hybridization assay conditions in a parallel or antiparallel orientation to form a stable structure having a double-stranded region.
  • the two constituent strands of this double-stranded structure sometimes called a hybrid, are held together by hydrogen bonds.
  • hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on single nucleic acid strands
  • base pairing can also form between bases which are not members of these "canonical” pairs.
  • Non-canonical base pairing is well-known in the art. See, e.g., R. L. P.
  • preferentially hybridize is meant that under stringent hybridization conditions, an amplification or detection probe oligomer can hybridize to its target nucleic acid to form stable oligo mcr: target hybrid, but not form a sufficient number of stable oligomernon-target hybrids.
  • Amplification and detection oligomers that preferentially hybridize to a target nucleic acid are useful to amplify and detect target nucleic acids, but not non-targeted organisms, especially phylogenetically closely related organisms.
  • the oligomer hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one having ordinary skill in the art to accurately amplify and/or detect the presence (or absence) of nucleic acid derived from the specified target as appropriate.
  • reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will decrease the degree or rate of hybridization of the
  • oligonucleotide to its target region.
  • inclusion of one or more non-complementary nucleosides or nucleobases may facilitate the ability of an oligonucleotide to discriminate against nontarget organisms.
  • Preferential hybridization can be measured using techniques known in the art and described herein, such as in the examples provided below.
  • there is at least a 10-fold difference between target and non-target hybridization signals in a test sample at least a 100- fold difference, or at least a 1,000-fold difference.
  • non-target hybridization signals in a test sample are no more than the background signal level.
  • stringent hybridization conditions conditions permitting an oligomer to preferentially hybridize to a target nucleic acid and not to nucleic acid derived from a closely related non-target nucleic acid. While the definition of stringent hybridization conditions does not vary, the actual reaction environment that can be used for stringent hybridization may vary depending upon factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and sequences of non-target nucleic acids that may be present in the test sample, and the target sequence.
  • Hybridization conditions include the temperature and the composition of the hybridization reagents or solutions.
  • Exemplary hybridization assay conditions for amplifying and/or detecting target nucleic acids derived from one or more serotypes of GBS with the oligomers of the present disclosure correspond to a temperature of about 60 °C when the salt concentration, such as a monovalent salt, e.g., KC1, is in the range of about 0.6-0.9 M.
  • the salt concentration such as a monovalent salt, e.g., KC1
  • Other acceptable stringent hybridization conditions are readily ascertained by those having ordinary skill in the art.
  • assay conditions conditions permitting stable hybridization of an oligonucleotide to a target nucleic acid. Assay conditions do not require preferential hybridization of the oligonucleotide to the target nucleic acid.
  • Label refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal.
  • Direct joining may use covalent bonds or non-covalent interactions (e.g. , hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordination complex formation) whereas indirect joining may use abridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s), which amplify a detectable signal.
  • Any detectable moiety may be used, e.g. , radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate, reactive group, chromophore such as a dye or particle (e.g. , latex or metal bead) that imparts a detectable color, luminescent compound (e.g., bioluminescent, phosphorescent, or
  • fluorophores include those that absorb light in the range of about 495 to 650 nm and emit light in the range of about 520 to 670 nm, which include those known as FAMTM, TETTM, CAL FLUORTM (Orange or Red), and QUASARTM compounds. Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to diminish background fluorescence. Such quenchers are well known in the art and include, e.g., BLACK HOLE QUENCHERTM (or BHQTM) or TAMRATM compounds. Particular embodiments include a
  • homogeneous detectable label that is detectable in a homogeneous system in which bound labeled probe in a mixture exhibits a detectable change compared to unbound labeled probe, which allows the label to be detected without physically removing hybridized from unhybridized labeled probe (e.g.,
  • Particular homogeneous detectable labels include chemiluminescent compounds, including acridinium ester ("AE") compounds, such as standard AE or AE derivatives which are well known (US Pat. Nos. 5,656,207, 5,658,737, and 5,639,604).
  • AE acridinium ester
  • Methods of synthesizing labels, attaching labels to nucleic acid, and detecting signals from labels are well known (e.g., Sambrook et al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Chapt. 10, and US Pat. Nos.
  • Particular methods of linking an AE compound to a nucleic acid are known (e.g., US Pat. No. 5,585,481 and US Pat. No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example 8).
  • Particular AE labeling positions are a probe's central region and near a region of A/T base pairs, at a probe's 3' or 5' terminus, or at or near a mismatch site with a known sequence that is the probe should not detect compared to the desired target sequence.
  • detectably labeled probes include TaqManTM probes, molecular torches, and molecular beacons.
  • TaqManTM probes include a donor and acceptor label wherein fluorescence is detected upon enzymatically degrading the probe during amplification in order to release the fluorophore from the presence of the quencher.
  • Molecular torches and beacons exist in open and closed configurations wherein the closed configuration quenches the fluorophore and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to target opens the otherwise closed probes.
  • Sequences are "sufficiently complementary” if they allow stable hybridization of two nucleic acid sequences, e.g. , stable hybrids of probe and target sequences, although the sequences need not be completely complementary. That is, a "sufficiently complementary" sequence that hybridizes to another sequence by hydrogen bonding between a subset series of complementary nucleotides by using standard base pairing (e.g., G:C, A:T, or A:U), although the two sequences may contain one or more residues (including abasic positions) that are not complementary so long as the entire sequences in appropriate hybridization conditions to form a stable hybridization complex. Sufficiently complementary sequences may be at least about 80%, at least about 90%, or completely complementary in the sequences that hybridize together. Appropriate hybridization conditions are well-known to those skilled in the art, can be predicted based on sequence composition, or can be determined empirically by using routine testing (e.g., Sambrook el al, Molecular Cloning, A
  • a "non-extendable" oligomer includes a blocking moiety at or near its 3 '-terminus to prevent extension.
  • a blocking group near the 3' end is in some embodiments within five residues of the 3' end and is sufficiently large to limit binding of a polymerase to the oligomer, and other embodiments contain a blocking group covalently attached to the 3' terminus.
  • Many different chemical groups may be used to block the 3' end, e.g., alkyl groups, non-nucleotide linkers, alkane- diol dideoxynucleotide residues, and cordycepin.
  • blocking moieties include a 3'- deoxy nucleotide (e.g., a 2',3'-dideoxy nucleotide); a 3'-phosphorylated nucleotide; a fluorophore, quencher, or other label that interferes with extension; an inverted nucleotide (e.g. , linked to the preceding nucleotide through a 3'-to-3' phosphodiester, optionally with an exposed 5'-OH or phosphate); or a protein or peptide joined to the oligonucleotide so as to prevent further extension of a nascent nucleic acid chain by a polymerase.
  • a 3'- deoxy nucleotide e.g., a 2',3'-dideoxy nucleotide
  • a 3'-phosphorylated nucleotide e.g., a fluorophore, quencher, or other label that interferes with extension
  • a non-extendable oligonucleotide of the present disclosure may be at least 10 bases in length, and may be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length.
  • Non-extendable oligonucleotides that comprise a detectable label can be used as probes.
  • sequence of SEQ ID NO: X refer to the base sequence of the corresponding sequence listing entry and do not require identity of the backbone (e.g., RNA, 2'-0-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues) unless the context clearly dictates otherwise.
  • backbone e.g., RNA, 2'-0-Me RNA, or DNA
  • base modifications e.g., methylation of cytosine residues
  • sample preparation refers to any steps or method that treats a sample for subsequent amplification and/or detection of GBS nucleic acids present in the sample. Samples may be complex mixtures of components of which the target nucleic acid is a minority component.
  • Sample preparation may include any known method of concentrating components, such as microbes or nucleic acids, from a larger sample volume, such as by filtration of airborne or waterborne particles from a larger volume sample or by isolation of microbes from a sample by using standard microbiology methods.
  • Sample preparation may include physical disruption and/or chemical lysis of cellular components to release intracellular components into a substantially aqueous or organic phase and removal of debris, such as by using filtration, centrifugation or adsorption.
  • Sample preparation may include use of a nucleic acid oligonucleotide that selectively or non-specifically capture a target nucleic acid and separate it from other sample components (e.g., as described in US Patent No.
  • Sample components include target nucleic acids usually in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids.
  • target nucleic acids usually in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids.
  • Separating or purifying does not connote any degree of purification. Typically, separating or purifying removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components.
  • non-linear surfactant means a surfactant having a branched chain structure.
  • a non-linear surfactant may include one or more ring structures, which may be, for example, in a principal chain and/or in one or more branch chains.
  • Exemplary non-linear surfactants include polysorbate 20, polysorbate 40, polysorbate 60, and digitonin.
  • a non-linear surfactant is non-ionic.
  • specificity in the context of an amplification and/or detection system, is used herein to refer to the characteristic of the system which describes its ability to distinguish between target and non-target sequences dependent on sequence and assay conditions.
  • specificity generally refers to the ratio of the number of specific amplicons produced to the number of side-products (e.g., the signal-to-noise ratio).
  • detection specifically refers to the ratio of signal produced from target nucleic acids to signal produced from non-target nucleic acids.
  • sensitivity is used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantitated.
  • the sensitivity of an amplification reaction is generally a measure of the smallest copy number of the target nucleic acid that can be reliably detected in the amplification system, and will depend, for example, on the detection assay being employed, and the specificity of the amplification reaction, e.g., the ratio of specific amplicons to side-products.
  • the present invention provides compositions, kits, and methods for amplifying and detecting Group B Streptococcus (GBS; Streptococcus agalactiae) nucleic acid from a sample.
  • the samples are biological samples.
  • the compositions, kits, and methods provide oligonucleotide sequences that recognize target sequences of the GBS genome, including target sequences of GBS serotypes la, lb, Ic, II, III, IV, V, VI, VII, VIII, and IX, or their complementary sequences.
  • oligonucleotides may be used as amplification oligonucleotides, which may include primers, promoter primers, blocked oligonucleotides, and promoter provider oligonucleotides, whose functions have been described previously (see, e.g., US Patent Nos. 4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and 7,374,885; each incorporated by reference herein).
  • Other oligonucleotides may be used as probes for detecting amplified sequences of GBS, or for capture of GBS target nucleic acid.
  • the methods provide for the sensitive and specific detection of GBS nucleic acids.
  • the methods include performing a nucleic acid amplification of an GBS target region and detecting the amplified product by, for example, specifically hybridizing the amplified product with a nucleic acid detection probe that provides a signal to indicate the presence of GBS in the sample.
  • the amplification step includes contacting the sample with one or more amplification oligomers specific for a target sequence in a GBS target nucleic acid to produce an amplified product if GBS nucleic acid is present in the sample.
  • Amplification synthesizes additional copies of the target sequence or its complement by using at least one nucleic acid polymerase and an amplification oligomer to produce the copies from a template strand (e.g., by extending the sequence from a primer using the template strand).
  • One embodiment for detecting the amplified product uses a hybridizing step that includes contacting the amplified product with at least one detection probe oligomer specific for a sequence amplified by the selected amplification oligomers, e.g. , a sequence contained in the target sequence flanked by a pair of selected amplification oligomers.
  • compositions of the instant invention are configured to specifically hybridize to nucleic acid of all GBS serotypes la, lb, Ic, II, III, IV, V, VI, VII, VIII, and IX with minimal cross-reactivity to other, non-GBS nucleic acids suspected of being in a sample ( e.g ., other bacterial pathogens).
  • compositions of the invention further allow detection of sequences on a non-hemolytic strain of GBS.
  • the compositions of the instant invention are configured to specifically hybridize to GBS nucleic acid with minimal cross-reactivity to one or more non-GBS pathogens listed in any of Tables 9-11, 15, 16, 20, and 22 ( see Examples, infra).
  • the compositions of the instant invention are part of a multiplex system that further includes components and methods for detecting one of more of these non-GBS pathogens.
  • a composition comprising at least two amplification oligomers for determining the presence or absence of GBS in a sample.
  • the composition includes at least two amplification oligomers for amplifying a target region of a GBS target nucleic acid corresponding to the sequence of SEQ ID NO: 1 ( SIP gene) or SEQ ID NO:2 ( CFB gene).
  • At least one amplification oligomer comprises a target-hybridizing sequence in the sense orientation ("sense THS”) and at least one amplification oligomer comprises a target-hybridizing sequence in the antisense orientation (“antisense THS”), where the sense THS and antisense THS are each configured to specifically hybridize to a GBS target sequence corresponding to a sequence contained within SEQ ID NO: 1 or SEQ ID NO: 2 and where the target-hybridizing sequences are selected such that the GBS sequence targeted by antisense THS is situated downstream of the GBS sequence targeted by the sense THS (i.e.. the at least two amplification oligomers are situated such that they flank the target region to be amplified).
  • sense THS and antisense THS are each configured to specifically hybridize to a GBS target sequence corresponding to a sequence contained within SEQ ID NO: 1 or SEQ ID NO: 2 and where the target-hybridizing sequences are selected such that the GBS sequence targeted by
  • a composition includes (i) a .S7/ J -spccific amplification oligomer comprising a SZP-specific target-hybridizing sequence substantially corresponding to, or identical to, the sequence shown in SEQ ID NOG, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8, or the complement thereof or an RNA equivalent or DNA/RNA chimeric thereof.
  • a composition includes (ii) a ⁇ ⁇ Vi-spccific amplification oligomer comprising a ⁇ ⁇ Vi-spccific target- hybridizing sequence that is from about 17 to about 24 contiguous nucleotides and substantially corresponding to, or identical to, a sequence that is contained in the sequence of SEQ ID NO:26, or the complement thereof or an RNA equivalent or DNA/RNA chimeric thereof; in some such embodiments, the C73 ⁇ 4-specific target-hybridizing sequence includes a sequence that substantially corresponds to, or is identical to, the sequence of SEQ ID NO:28 or SEQ ID NO:27, or the complement thereof or an RNA equivalent or DNA/RNA chimeric thereof (e.g.
  • a composition includes (iii) a CE //-specific amplification oligomer comprising a CE //-specific target- hybridizing sequence substantially corresponding to, or identical to, the sequence shown in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:20, or SEQ ID NO:21, or the complement thereof or an RNA equivalent or DNA/RNA chimeric thereof.
  • the oligomer combination includes at least one an amplification oligomer comprising an .S/B-spccific or CEB- specific target-hybridizing sequence of the opposite polarity (sense vs. antisense or vice versa) as the target-hybridizing sequence of the oligomer of (i), (ii), or (iii), such that at least two amplification oligomers flank a target region to be amplified.
  • the composition is provided as an aqueous or dried formulation for amplification of GBS nucleic acid, or a reaction mixture comprising or reconstituted from such a formulation.
  • a composition for determining the presence or absence of GBS in a sample includes (1) at least one amplification oligomer comprising a .S/B-spccific or CEB-specific target-hybridizing region substantially corresponding to at least one sense oligomer sequence depicted in Table 1 below, and (2) at least one amplification oligomer comprising a .S/B-spccific or CEB-specific target hybridizing region substantially corresponding to at least one antisense oligomer sequence depicted in Table 1.
  • the composition includes a first .S/B-spccific amplification oligomer and a first CEB-specific amplification oligomer of (1) above and a second .S/B-spccific and second CEB-specific amplification oligomer of (2) above.
  • the sense and/or antisense target- hybridizing sequence(s) of an amplification oligomer combination comprises or consists of the sense and/or antisense sequence(s) selected from Table 1.
  • a composition for determining the presence or absence of GBS in a sample as described herein further comprises at least one detection probe oligomer configured to specifically hybridize to a GBS SIP or CFB target sequence that is amplifiable using the first and second amplification oligomers (e.g. , an SIP or CFB target sequence contained within SEQ ID NO: 1 or SEQ ID NO:2, or the complement thereof, that is flanked by the target-hybridizing sequences of the first and second amplification oligomers).
  • a detection probe oligomer configured to specifically hybridize to a GBS SIP or CFB target sequence that is amplifiable using the first and second amplification oligomers (e.g. , an SIP or CFB target sequence contained within SEQ ID NO: 1 or SEQ ID NO:2, or the complement thereof, that is flanked by the target-hybridizing sequences of the first and second amplification oligomers).
  • Particularly suitable .S'/P-spccific detection probe oligomers include, for example, oligomers comprising a SZP-specific target-hybridizing sequence substantially corresponding to, or identical to, the sequence shown in SEQ ID NO:9 or SEQ ID NO:9
  • C73 ⁇ 4-specific detection probe oligomers include, for example, oligomers comprising a CFB- specific target-hybridizing sequence substantially corresponding to, or identical to, the sequence shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or the complement thereof or an RNA equivalent or DNA/RNA chimeric thereof.
  • a detection probe oligomer may contain a 2'-methoxy backbone at one or more linkages in the nucleic acid backbone.
  • a composition includes at least two detection probe oligomers.
  • a detection probe oligomer is provided in an aqueous or dried formulation for detection of GBS nucleic acid, or a reaction mixture comprising or reconstituted from such a formulation
  • a detection probe oligomer in accordance with the present invention further includes a label.
  • Particularly suitable labels include compounds that emit a detectable light signal, e.g., fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label, may be present on a particular probe, or detection may rely on using a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (see, e.g., US Pat. Nos. 6,180,340 and 6,350,579, each incorporated by reference herein).
  • Labels may be attached to a probe by various means including covalent linkages, chelation, and ionic interactions, but preferably the label is covalently attached.
  • a detection probe has an attached chemiluminescent label such as, e.g., an acridinium ester (AE) compound (see, e.g., US Patent Nos. 5,185,439;
  • AE acridinium ester
  • a label such as, e.g., a fluorescent or chemiluminescent label, is typically attached to the probe by a non-nucleotide linker (see, e.g., US Patent Nos. 5,585,481; 5,656,744; and 5,639,604, particularly at column 10, line 6 to column 11, line 3, and Example 8; each incorporated by reference herein).
  • a probe (e.g., comprising a fluorescent label) further includes a second label that interacts with the first label.
  • the second label can be a quencher.
  • Detection probes comprising both a fluorescent label and a quencher, a combination are particularly useful in fluorescence resonance energy transfer (FRET) assays.
  • FRET fluorescence resonance energy transfer
  • Specific variations of such detection probes include, e.g., a TaqManTM detection probe (Roche Molecular Diagnostics) and a "molecular beacon" (see, e.g., Tyagi et al, Nature Biotechnol. 16:49-53, 1998; US Patent Nos. 5,118,801 and 5,312,728; each incorporated by reference herein).
  • TaqManTM probes (or similar dual-labeled linear probes comprising both a fluorescent label and a quencher), can be used in assays where hybridization of the probe to a target or amplicon followed by nucleolysis by a polymerase comprising 5 '-3' exonuclease activity results in liberation of the fluorescent label and thereby increased fluorescence, or fluorescence independent of the interaction with the second label.
  • a detection probe exhibiting at least some degree of selfcomplementarity are used to facilitate detection of probe :target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection.
  • detection probes include, for example, probes that form conformations held by intramolecular hybridization, such as conformations generally referred to as hairpins.
  • Suitable hairpin probes include a "molecular torch” (see, e.g., U.S. Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945) and a "molecular beacon" (see, e.g., U.S. Pat. No.
  • Molecular torches include distinct regions of self-complementarity (coined “the target binding domain” and “the target closing domain") which are connected by a joining region (e.g., a -(CFFCFhOb- linker) and which hybridize to one another under predetermined hybridization assay conditions.
  • a joining region e.g., a -(CFFCFhOb- linker
  • the two complementary regions which may be fully or partially complementary
  • the target binding domain favors hybridization to the target sequence over the target closing domain.
  • the target binding domain and the target closing domain of a molecular torch include interacting labels (e.g., fluorescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized as opposed to when the molecular torch is hybridized to a target nucleic acid, thereby permitting detection of proberiarget duplexes in a test sample in the presence of unhybridized probe having a viable label associated therewith.
  • a detection probe is a linear oligomer that does not substantially form conformations held by intramolecular bonds.
  • a linear detection probe oligomer includes a chemiluminescent compound as the label (e.g.
  • a linear detection probe oligomer includes a fluorophore as the label.
  • the oligomer further includes a quenching moiety (e.g., a TaqMan probe).
  • Examples of interacting donor/acceptor label pairs that may be used in connection with the disclosure, making no attempt to distinguish FRET from non-FRET pairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluororescein, EDANS/DABCYL,
  • Non-fluorescent acceptors such as DABCYL and the QSY7 dyes advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e.. non-sensitized) acceptor excitation.
  • exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and the CY dyes (such as CY5).
  • exemplary quencher moieties that can be used as another member of a donor-acceptor pair include DABCYL and the BLACK HOLE QUENCHER moieties which are available from Biosearch Technologies, Inc., (Novato, Calif.).
  • a labeled oligomer (e.g. , a detection probe) is non-extendable.
  • the labeled oligomer can be rendered non-extendable by 3 '-phosphorylation, having a 3'- terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), having a 3'-terminal inverted nucleotide (e.g.
  • the last nucleotide is inverted such that it is joined to the penultimate nucleotide by a 3' to 3' phosphodiester linkage or analog thereof, such as a phosphorothioate), or having an attached fluorophore, quencher, or other label that interferes with extension (possibly but not necessarily attached via the 3' position of the terminal nucleotide).
  • the 3'- terminal nucleotide is not methylated.
  • compositions comprising one or more detection probe oligomers as described herein.
  • the present invention provides methods utilizing an oligomer or oligomer combination as described herein. Any method disclosed herein is also to be understood as a disclosure of corresponding uses of materials involved in the method directed to the purpose of the method. Any of the oligomers comprising a GBS SIP- or CFB-target-hybridizing sequence and any combinations (e.g., kits and compositions) comprising such an oligomer are to be understood as also disclosed for use in detecting or quantifying GBS, and for use in the preparation of a composition for detecting or quantifying GBS.
  • methods may comprise one or more of the following components: target capture, in which GBS nucleic acid (e.g., from a sample, such as a clinical sample) is annealed to a capture oligomer; isolation, e.g., washing, to remove material not associated with a capture oligomer; amplification; and amplicon detection, e.g., amplicon quantification, which may be performed in real time with amplification.
  • Certain embodiments involve each of the foregoing steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two components listed adjacently above, e.g., washing and amplification, or amplification and detection.
  • the present invention provides a method for determining the presence or absence of Group B Streptococcus (GBS) in a sample using an oligomer combination as described herein.
  • a method generally includes (1) contacting the sample with at least two amplification oligomers for amplifying a GBS SIP or CFB nucleic acid target region corresponding to a SIP or CFB target nucleic acid, where the at least two amplification oligomers are as described above; (2) performing an in vitro nucleic acid amplification reaction, where any GBS SIP or CFB target nucleic acid present in the sample is used as a template for generating an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of GBS in the sample.
  • GBS Group B Streptococcus
  • a detection method in accordance with the present invention typically further includes the step of obtaining the sample to be contacted with the at least two amplification oligomers.
  • "obtaining" a sample to be used in steps (l)-(3) includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and/or retrieving the sample from a location (e.g. , from storage or other depository) within a facility where one or more steps of the method are performed.
  • Amplifying a GBS target sequence utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified.
  • the target region to be amplified is a GBS SIP target region substantially corresponding to SEQ ID NO: 1 from about nucleotide position 56 to about nucleotide position 189 or from about nucleotide position 349 to about nucleotide position 489.
  • Particularly suitable oligomer combinations for amplification of these GBS SIP target regions are described herein.
  • an amplification oligomer combination for amplifying a SIP target region includes first and second .S7/ J -spccific amplification oligomers comprising, respectively, (A) a first STP-specific target-hybridizing sequence that is SEQ ID N0:3 or a sequence substantially corresponding to SEQ ID NO:3, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second .S/B-spccific target- hybridizing sequence that is SEQ ID NO:4 or a sequence substantially corresponding to SEQ ID NO:4, or an RNA equivalent or DNA/RNA chimeric thereof.
  • an amplification oligomer combination for amplifying a SIP target region includes first and second .S/B-spccific amplification oligomers comprising, respectively, (A) a first STP-specific target-hybridizing sequence that is SEQ ID NO:7 or a sequence substantially corresponding to SEQ ID NO:7, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second STP-specific target-hybridizing sequence that is SEQ ID NO:8 or a sequence substantially corresponding to SEQ ID NO:8, or an RNA equivalent or DNA/RNA chimeric thereof.
  • the target region to be amplified is a GBS CFB target region substantially corresponding to SEQ ID NO:2 from about nucleotide position 38 to about nucleotide position 151, from about nucleotide position 22 to about nucleotide position 151, from about nucleotide position 192 to about nucleotide position 329, or from about nucleotide position 585 to about nucleotide position 716.
  • GBS CFB target region substantially corresponding to SEQ ID NO:2 from about nucleotide position 38 to about nucleotide position 151, from about nucleotide position 22 to about nucleotide position 151, from about nucleotide position 192 to about nucleotide position 329, or from about nucleotide position 585 to about nucleotide position 716.
  • Particularly suitable oligomer combinations for amplification of these GBS CFB target regions are described herein.
  • an amplification oligomer combination for amplifying a CFB target region includes first and second CEB-spccific amplification oligomers comprising, respectively, (A) a first CEB-specific target-hybridizing sequence that is from about 17 to about 24 contiguous nucleotides and substantially corresponding to, or identical to, a sequence that is contained in the sequence of SEQ ID NO:26, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second CEB-specific target-hybridizing sequence that is SEQ ID NO: 13 or SEQ ID NO: 15 or a sequence substantially corresponding to SEQ ID NO: 13 or SEQ ID NO: 15, or an RNA equivalent or DNA/RNA chimeric thereof; in more specific variations of such a first CEB-specific target-hybridizing sequence of (A), the CEB-specific target-hybridizing sequence is selected from (i) a sequence that substantially corresponds to, or is identical to, the sequence of
  • an amplification oligomer combination for amplifying a CEB target region includes first and second CEB-specific amplification oligomers comprising, respectively, (A) a first CEB-specific target- hybridizing sequence that is SEQ ID NO: 16 or a sequence substantially corresponding to SEQ ID NO: 16, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second CEB-specific target- hybridizing sequence that is SEQ ID NO: 17 or a sequence substantially corresponding to SEQ ID NO: 17, or an RNA equivalent or DNA/RNA chimeric thereof.
  • an amplification oligomer combination for amplifying a CFB target region includes first and second 77 ⁇ //-specific amplification oligomers comprising, respectively, (A) a first C73 ⁇ 4-specific target- hybridizing sequence that is SEQ ID NO: 18 or a sequence substantially corresponding to SEQ ID NO: 18, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second ( 7' //-specific target- hybridizing sequence that is SEQ ID NO: 15 or a sequence substantially corresponding to SEQ ID NO: 15, or an RNA equivalent or DNA/RNA chimeric thereof.
  • an amplification oligomer combination for amplifying a CFB target region includes first and second 77' //-sped fie amplification oligomers comprising, respectively, (A) a first C73 ⁇ 4-specific target- hybridizing sequence that is SEQ ID NO:20 or a sequence substantially corresponding to SEQ ID NO:20, or an RNA equivalent or DNA/RNA chimeric thereof, and (B) a second 77' //-specific target- hybridizing sequence that is SEQ ID NO:21 or a sequence substantially corresponding to SEQ ID NO:21, or an RNA equivalent or DNA/RNA chimeric thereof.
  • a detection method in accordance with the present disclosure can further include the step of obtaining the sample to be subjected to subsequent steps of the method.
  • "obtaining" a sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and/or retrieving the sample from a location (e.g., from storage or other depository) within a facility where one or more steps of the method are performed.
  • the method further includes purifying the GBS target nucleic acid from other components in the sample, e.g., before an amplification, such as before a capture step.
  • purification may include methods of separating and/or concentrating organisms contained in a sample from other sample components, or removing or degrading non-nucleic acid sample components, e.g. , protein, carbohydrate, salt, lipid, etc.
  • DNA in the sample is degraded, e.g., with DNase, and optionally removing or inactivating the DNase or removing degraded DNA.
  • purifying the target nucleic acid includes capturing the target nucleic acid to specifically or non-specifically separate the target nucleic acid from other sample components.
  • Non-specific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adherence of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture that contains GBS nucleic acid and other sample components
  • Target capture typically occurs in a solution phase mixture that contains one or more capture probe oligomers that hybridize to the GBS SIP or CFB target sequence under hybridizing conditions.
  • the GBS-target:capture-probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to an immobilized probe.
  • Certain embodiments use a particulate solid support, such as paramagnetic beads.
  • Isolation can follow capture, where, for example, the complex on the solid support is separated from other sample components. Isolation can be accomplished by any appropriate technique, e.g. , washing a support associated with the GBS SIP or CFB target-sequence one or more times (e.g. , two or three times) to remove other sample components and/or unbound oligomer. In embodiments using a particulate solid support, such as paramagnetic beads, particles associated with the GBS target may be suspended in a washing solution and retrieved from the washing solution, in some embodiments by using magnetic attraction. To limit the number of handling steps, the GBS SIP or CFB target nucleic acid may be amplified by simply mixing the GBS target sequence in the complex on the support with amplification oligomers and proceeding with amplification steps.
  • Exponentially amplifying a GBS target sequence utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified.
  • at least first and second oligomers as described herein are provided.
  • a plurality of pairs of oligomers is provided; in some such variations, a plurality of oligomer pairs comprises oligomer pairs configured to hybridize to at least two GBS target nucleic acids (e.g. , at least one oligomer pair configured to hybridized to a SIP target nucleic acid and at least one oligomer pair configured to hybridize to a CFB target nucleic acid).
  • the amplification reaction can be cycled or isothermal.
  • Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand-displacement amplification
  • TMA transcription-mediated or transcription-associated amplification
  • a detection step may be performed using any of a variety of known techniques to detect a signal specifically associated with the amplified target sequence, such as, e.g. , by hybridizing the amplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (including from label released from the probe following hybridization in some embodiments).
  • the labeled probe comprises a second moiety, such as a quencher or other moiety that interacts with the first label, as discussed above.
  • the detection step may also provide additional information on the amplified sequence, such as, e.g. , all or a portion of its nucleic acid base sequence.
  • Detection may be performed after the amplification reaction is completed, or may be performed simultaneously with amplifying the target region, e.g., in real time.
  • the detection step allows homogeneous detection, e.g. , detection of the hybridized probe without removal of unhybridized probe from the mixture (see. e.g., U.S. Patent Nos. 5,639,604 and 5,283,174).
  • the nucleic acids are associated with a surface that results in a physical change, such as a detectable electrical change. Amplified nucleic acids may be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g.
  • nucleic acid detection probes that are configured to specifically hybridize to a sequence in the amplified product and detecting the presence of the probe:product complex, or by using a complex of probes that may amplify the detectable signal associated with the amplified products (e.g., U.S. Patent Nos. 5,424,413; 5,451,503; and 5,849,481; each incorporated by reference herein).
  • Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal that indicates the presence of the target nucleic acid in the sample.
  • the amplified product will contain a target sequence in or complementary to a sequence in the GBS SIP or CFB gene, and a probe will bind directly or indirectly to a sequence contained in the amplified product to indicate the presence of GBS nucleic acid in the tested sample.
  • a linear detection probe may be used to provide a signal to indicate hybridization of the probe to the amplified product.
  • detection uses a luminescentally labeled probe that hybridizes to target nucleic acid. Luminescent label is then hydrolyzed from non- hybridized probe. Detection is performed by chemiluminescence using a luminometer. (see, e.g., International Patent Application Pub. No. WO 89/002476, incorporated by reference herein).
  • the detection probe may be a hairpin probe such as, for example, a molecular beacon, molecular torch, or hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to amplified product (e.g., a dual-labeled hairpin probe comprising both a fluorescent label and a quenching moiety).
  • the detection probe is a linear oligomer such as, e.g. , an oligomer labeled with both a fluorophore and a quenching moiety (e.g. , a TaqMan probe).
  • Such probes may comprise target- hybridizing sequences and non-target-hybridizing sequences.
  • Various forms of such probes have been described previously (see, e.g., US Patent Nos. 5,210,015; 5,487,972; 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1; each incorporated by reference herein).
  • Assays for detection of the GBS nucleic acid may optionally include a non-GBS internal control (IC) nucleic acid that is amplified and detected in the same assay reaction mixtures by using amplification and detection oligomers specific for the IC sequence.
  • IC nucleic acid sequences can be, e.g., a DNA plasmid, an RNA template sequence (e.g., an in vitro transcript), or a synthetic nucleic acid that is spiked into a sample.
  • the IC nucleic acid sequence may be a cellular component, which may be from exogenous cellular sources or endogenous cellular sources relative to the specimen.
  • an internal control nucleic acid is co-amplified with the GBS nucleic acid in the amplification reaction mixtures.
  • the internal control amplification product and the GBS target sequence amplification product can be detected independently.
  • amplification and detection of a signal from an amplified IC sequence demonstrates that the assay reagents, conditions, and performance of assay steps were properly used in the assay if no signal is obtained for the intended target GBS nucleic acid (e.g. , samples that test negative for GBS).
  • An IC may also be used as an internal calibrator for the assay when a quantitative result is desired, i.e., the signal obtained from the IC amplification and detection is used to set a parameter used in an algorithm for quantitating the amount of GBS nucleic acid in a sample based on the signal obtained for an amplified GBS target sequence.
  • ICs are also useful for monitoring the integrity of one or more steps in an assay.
  • the primers and probe for the IC target sequence are configured and synthesized by using any well-known method provided that the primers and probe function for amplification of the IC target sequence and detection of the amplified IC sequence using substantially the same assay conditions used to amplify and detect the GBS target sequence.
  • a target capture probe specific for the IC target be included in the assay in the target capture step so that the IC is treated in the assay in a manner analogous to that for the intended GBS analyte in all of the assay steps.
  • a formulation for determining the presence or absence of GBS in a sample.
  • a formulation is an aqueous formulation comprising (1) at least two .S'/C-spccific or CAB-specific amplification oligomers for amplification of a SIP or CFB target region as described herein and (2) an organic buffer.
  • An aqueous formulation for amplification of a GBS nucleic acid may include one or more additional components such as, e.g., a DNA polymerase enzyme, a reverse transcriptase enzyme, or a detection probe oligomer.
  • a formulation is an aqueous formulation comprising (1) a .SYC-spccific and/or CFB- specific detection probe oligomer as described herein and (2) on organic buffer.
  • An aqueous formulation for comprising or more detection probe oligomers may include one or more additional components such as, e.g., a surfactant, a DNA polymerase enzyme, a reverse transcriptase enzyme, or at least one amplification oligomer.
  • surfactants include, for example, polyethylene glycol mono [4-(l,l,3,3-tetramethylbutyl) phenyl] ether and polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, or polysorbate 60).
  • a surfactant in an aqueous detection probe formulation is a non-linear surfactant such as, for example, a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 40, or polysorbate 60) or digitonin.
  • An aqueous formulation as above for amplification or detection of GBS nucleic acid may further include a bulking agent such as, e.g., trehalose, raffinose, or a combination thereof.
  • a bulking agent such as, e.g., trehalose, raffinose, or a combination thereof.
  • an aqueous formulation as above contains at inorganic salt such as, e.g. , magnesium, potassium, or sodium; in some such variations, the concentration of the inorganic salt is 4 mM or less.
  • a particularly suitable organic buffer for an aqueous formulation as above is Tris (2 -amino-2 - (hydroxymethyl)- 1 ,3 -propanediol).
  • an aqueous formulation as described herein may be aliquoted into, e.g., vials, ampules, or other containers and dried (e.g., lyophilized) according to procedures known in the art.
  • the dried product typically appears as a powder or cake.
  • the containers are then sealed.
  • Methods of preparing such dried formulations from the aqueous formulation, as well as the dried formulations prepared by such methods, are additional aspects of the present invention.
  • the present invention provides a dried formulation that enables reconstitution into an aqueous formulation as described herein.
  • Dried formulations for amplification or detection of GBS nucleic acid typically contain, in addition to one or more amplification oligomers and/or detection probes as described herein, a bulking agent such as, e.g. , trehalose, raffinose, or a combination thereof.
  • a bulking agent such as, e.g. , trehalose, raffinose, or a combination thereof.
  • the percent mass of the inorganic salt to the mass of the dried formulation is 0.249% or less, 0.222% or less, or 0.195% or less.
  • a reaction mixture for determining the presence or absence of a GBS target nucleic acid in a sample includes one or both of (1) an oligomer combination as described herein for amplification of a GBS SIP and/or CFB target nucleic acid and (2) one or more detection probe oligomers as described herein for determining the presence or absence of a GBS SIP and/or CFB amplification product.
  • the reaction mixture may further include a number of optional components such as, for example, capture probes, e.g., poly-(k) capture probes as described in US 2013/0209992, which is incorporated herein by reference.
  • the reaction mixture will typically include other reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and/or ATP, CTP, GTP and UTP), and/or enzymes (e.g., a thermostable DNA polymerase, or reverse transcriptase and/or RNA polymerase), and will typically include test sample components, in which a GBS target nucleic acid may or may not be present.
  • nucleotide triphosphates e.g., dATP, dCTP, dGTP, and dTTP
  • enzymes e.g., a thermostable DNA polymerase, or reverse transcriptase and/or RNA polymerase
  • a reaction mixture may include amplification oligomers for only one target region of a GBS genome, or it may include amplification oligomers for multiple GBS target regions (e.g., both a SIP target region and a CFB target region).
  • selection of amplification oligomers and detection probe oligomers for a reaction mixture are linked by a common target region (/. e. , the reaction mixture will include a probe that binds to a sequence amplifiable by an amplification oligomer combination of the reaction mixture).
  • a reaction mixture comprises an aqueous formulation as described above.
  • a reaction mixture is reconstituted with water or an organic buffer from a dried formulation as described above.
  • kits for practicing the methods as described herein include one or both of (1) an oligomer combination as described herein for amplification of a GBS SIP and/or CFB target nucleic acid and (2) one or more detection probe oligomers as described herein for determining the presence or absence of a GBS SIP and/or CFB amplification product.
  • any oligomer combination described herein is present in the kit.
  • the kits may further include a number of optional components such as, for example, capture probes, e.g., poly-(k) capture probes as described in US 2013/0209992.
  • kits include reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP; and/or ATP, CTP, GTP and UTP), and/or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase and/or RNA polymerase). Oligomers as described herein may be packaged in a variety of different embodiments, and those skilled in the art will appreciate that the disclosure embraces many different kit configurations.
  • nucleotide triphosphates e.g., dATP, dCTP, dGTP, dTTP; and/or ATP, CTP, GTP and UTP
  • enzymes e.g., a thermostable DNA polymerase, or a reverse transcriptase and/or RNA polymerase.
  • Oligomers as described herein may be
  • a kit may include amplification oligomers for only one target region of a GBS genome, or it may include amplification oligomers for multiple GBS target regions (e.g. , both a SIP target region and a CFB target region).
  • a kit that includes a detection probe together with an amplification oligomer combination selection of amplification oligomers and detection probe oligomers for a kit are linked by a common target region (/. e. , the kit will include a probe that binds to a sequence amplifiable by an amplification oligomer combination of the kit).
  • the kit further includes a set of instructions for practicing methods in accordance with the present disclosure, where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof.
  • primer/probe combinations 3 and 17 were selected for further evaluation for sensitivity and specificity. These primer combinations were able to detect five (5) theoretical copies per PCR reaction at a Ct of 36-37.
  • the initial testing of the primers and probes was performed on the ABI 7500 FAST Real-Time PCR System after KINGFISHERTM extraction. Three different primer/probe concentrations were used: 600/200nM, 400/150nM and 300/100nM.
  • the IC oligos were tested in combination with the SIP oligos when a GBS target is present (serotype II and IV: strains obtained from the CHU de vide).
  • the second step of the IC testing was performed on the BioRad CFX-96 qPCR device, which is capable of detecting both Cy5 and QUASAR705. Initially, the QUASAR705 combination was tested in comparison to the Cy5 combination to define the final concentration of the primers and probes.
  • Table 11 shows specificity data obtained on ABI 7500 FAST system. All tested bacteria showed no interaction with the SIP primers and probes. The effectiveness of the PCR was evaluated by a positive control, which rendered positive signals.
  • primer/probe combination 3 targeting the GBS SIP gene only, was compared to that of both primer/probe combinations 3 and 17 (as a multiplex reaction), targeting both the SIP and CFB genes, respectively.
  • Different concentrations of GBS primer/probe were tested on a GBS strain spiked in Specimen Transport Medium (STM) at 3000 CFU/PCR, using ABI 7500 FAST Real-Time PCR System after extraction in the KINGFISHERTM system.
  • STM Specimen Transport Medium
  • the GBS strain serotype III was serially diluted in Lim Broth at eight concentrations (based on plating): 20,000.0, 10,000.0, 5,000.0, 2,500.0, 1,250.0, 625.0, 312.5 and 156.3 CFU/mL, starting from a previous GBS culture (stock solution in Specimen Transport Medium (STM)).
  • a panel of 124 organisms consisting of 104 bacterial, 12 viral, 4 yeast/fungi and 4 protozoa/parasite strains representing microorganisms commonly found in vaginal/anal flora or that are the same family/genus as GBS were selected for analytical specificity testing.
  • the analytical specificity panel is detailed in Table 22.
  • 14 were not available for testing at the time of the study.
  • the potential cross reactivity with the GBS assay primers and probes for those 14 unavailable organisms were assessed by BLAST analysis with no alignments identified.
  • Cross-reactivity ( exclusivity ): testing whether these organisms cross-react with the GBS assay primers and probes and induce a false positive result in the confirmed GBS negative samples;

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Abstract

L'invention concerne des oligomères d'acide nucléique, notamment des oligomères d'amplification et des sondes de détection, pour la détection du streptocoque du groupe B (GBS; Streptococcus agalactiae). L'invention concerne également des procédés d'amplification et de détection d'acides nucléiques spécifiques à l'aide des oligomères décrits, ainsi que des mélanges réactionnels et des kits correspondants.
PCT/IB2019/056814 2018-06-13 2019-08-09 Compositions et procédés de détection d'acide nucléique de streptocoque du groupe b WO2019239394A1 (fr)

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GB2100233.2A GB2590210B (en) 2018-06-13 2019-08-09 Compositions and methods for detecting group B Streptococcus nucleic acid
CA3103442A CA3103442A1 (fr) 2018-06-13 2019-08-09 Compositions et procedes de detection d'acide nucleique de streptocoque du groupe b
AU2019286648A AU2019286648B2 (en) 2018-06-13 2019-08-09 Compositions and methods for detecting group B Streptococcus nucleic acid
CH001571/2020A CH716454B1 (de) 2018-06-13 2019-08-09 Zusammensetzungen und Verfahren zum Nachweis der Nukleinsäure von Gruppe-B-Streptococcus.
CN201980051387.4A CN112654721A (zh) 2018-06-13 2019-08-09 用于检测b群链球菌核酸的组合物和方法
AU2023202451A AU2023202451B2 (en) 2018-06-13 2023-04-21 Compositions and methods for detecting group B Streptococcus nucleic acid
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FR3087205A1 (fr) * 2018-06-13 2020-04-17 Gen Probe Incorporated Compositions et procédés de détection d’acide nucléique à streptocoque de groupe B

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WO2024184165A1 (fr) * 2023-03-03 2024-09-12 F. Hoffmann-La Roche Ag Compositions et procédés pour la détection de streptococcus groupe b ( streptococcus agalactiae) et des déterminants du gène de résistance à la clindamycine

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